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(12) United States Patent (10) Patent No.: US 6,413,571 Bl Liu (45) Date of Patent: Jul. 2, 2002

(54) STEROL ESTERS OF CONJUGATED Abstract from DATABASE CHEMABS Online!. Chemical LINOLEIC ACIDS AND PROCESS FOR Abstracts Service, Columbus, OH, US; Kaoru, Kazuhisa et THEIR PRODUCTION al: "Conjugated linoleic acid esters as antioxidants, their manufacture, and antioxidants and food containing the (75) Inventor: Linsen Liu, Irvine, CA (US) esters" retrieved from STN Database accession No. 133:221697 XP 002167426 abstracted from JP 2000 247965 (73) Assignee: The Board of Regents for Oklahoma A, (Snow Brand Milk Products Co., Ltd., Japan) Sept. 12, State University, Stillwater, OK (US) 2000. ( *) Notice: Subject to any disclaimer, the term of this Abstract of Dyas, L. et al.: "Steryl fatty acyl esters in patent is extended or adjusted under 35 plants", Phytochemistry, Sept. 1993, v. 34(1) pp. 17-29. U.S.C. 154(b) by O days. Abstract of Field, F. et al.: "Effect of micellar beta-sitosterol (21) Appl. No.: 09/693,562 on cholesterol metabolism in CaCo-2 cells", Journal of Lipid Research, Feb. 1997, v. 38(2) pp. 348-360. (22) Filed: Oct. 20, 2000 Abstract of Gylling, H. Siimes, et al.: "Sitostanol ester Related U.S. Application Data margarine in dietary treatment of children with familial (60) Provisional application No. 60/160,894, filed on Oct. 21, hypercholesterolemia", Journal of Lipid Research, Aug. 1999. 1995, V. 36(8) pp. 1807-1812. (51) Int. Cl.7 ...... A23D 9/007 Abstract of Micich, T.J. et al.: "Polymer-supported (52) U.S. Cl...... 426/611; 552/544; 552/547; saponins: an approach to cholesterol removal from but­ 552/554; 554/174 teroil", Journal of Agricultural and Food Chemistry, Aug. (58) Field of Search ...... 426/611; 552/544, 1992, V. 40 (8) pp. 1321-1325. 552/545, 547,554; 514/170, 171, 182, 562; 554/174; 435/134, 135 Haumann, Barbara F.: "Conjugated linoleic acid offers research promise", Inform, 1996, pp. 152-153, 156-159, (56) References Cited vol. 7, No. 2. U.S. PATENT DOCUMENTS O'Shea, M. et al.: "Conjugated linoleic acid in bovine milk 5,502,045 A 3/1996 Miettinen et al. fat: a food-based approach to cancer chemoprevention", 5,554,646 A 9/1996 Cook et al. Trends in Food Science & Technology, 1998, pp. 192-196, 5,760,082 A 6/1998 Cook et al. vol. 9, Elsevier Science Ltd. 5,892,068 A 4/1999 Higgins, III 5,919,451 A 7/1999 Cook et al. "", Critical Reviews in Food Science and Nutri­ 6,031,118 A * 2/2000 van Amerongen et al. . 552/544 tion, 1999, pp. 275-283, vol. 39, No. 3, CRC Press LLC. 6,106,886 A * 8/2000 van Amerongen et al. . 426/611 6,123,979 A * 9/2000 Hepburn et al...... 426/611 Steinhart, Carol: "Conjugated LinoleicAcid the Good News 6,139,897 A * 10/2000 Goto et al...... 426/601 about Animal Fat", Journal of Chemical Education, 1996, p. 6,147,236 A * 11/2000 Higgins, III...... 552/554 A302, vol. 73, No. 12. 6,231,915 Bl * 5/2001 van Amerongen et al. . 426/611 2002/0010349 Al * 1/2002 Roden * cited by examiner FOREIGN PATENT DOCUMENTS

DE 197 50 422 C 1 11/1998 ...... C07J/9/00 Primary Examiner-Carolyn Paden EP 0 982 316 A 3/2000 ...... C07J/9/00 (74) Attorney, Agent, or Firm-Fellers, Snider, WO WO98/38206 * 9/1998 Blankenship, Bailey & Tippens, P.C. WO WO 99/56558 11/1999 OIBER PUBLICATIONS (57) ABSTRACT J. Agricultural and Food Chem 49(11)5210-5216. * Novel sterol/stanol esters of a conjugated fatty acid are Abstract from DATABASE BIOSIS Online!. BIO­ provided through the esterification or transesterification of a SCIENCES INFORMATION Service, Philadelphis, PA, sterol such as beta-sitosterol or a hydrogenated form thereof US; May 1999 (1999-05); Gylling Helena et al: "Choles­ (stanol). Such novel esters exhibit the combined properties terol reduction by different plant stanol mixtures and with normally possess by the sterol/stanol compound and the variable fat intake." Database access10n No. conjugated fatty acid and as such are excellent additives for PREV199900281083 XP002167425 abstracted from dietetic foods and supplements. Metabolism Clinical and Experimental, vol. 48, No. 5 May 1999, pp. 575-580. ISSN: 0026-0495. 10 Claims, No Drawings US 6,413,571 Bl 1 2 STEROL ESTERS OF CONJUGATED of a sterol or stanol with a conjugated fatty acid, such as LINOLEIC ACIDS AND PROCESS FOR CLA, there is provided a compound which provides the THEIR PRODUCTION advantages of both the conjugated fatty acid and the sterol or stanol. CLA is a liquid fatty acid with two conjugated CROSS REFERENCE TO RELATED 5 double bonds, therefore, it can reduce the melting point of APPLICATION sterols and stanols dramatically. Indeed, the beta-sitosterol This application claims the benefit of my copending U.S. ester of CLA is liquid at ambient temperature while the provisional application Serial No. 60/160,894, filed Oct. 21, current commercial products made of the fatty acids derived 1999. from vegetable oils are solid or semisolid. The sterol ester of 10 CLA also provides a product having lower total calories than BACKGROUND OF THE INVENTION the blended product that provides the same doses of sterol and CLA. Such new products thus offer the combined 1. Technical Field benefits of sterols/stanols as a cholesterol control agent and This invention relates to novel sterol esters of conjugated CLA as an anticarcinogen and fat reducing agent. Such linoleic acids and a process for the production of the same 15 esters can be used as a supplement or ingredient in foods. by esterification of sterols and stanols with a conjugated In accordance with another embodiment of the present, linoleic acid. sterol esters can be readily prepared through esterification of 2. Background sterol or stanol with the conjugated fatty acid or by trans­ It is known that the addition of plant sterol () esterification of sterol or stanol with of the conjugated fatty to diets will reduce serum cholesterol levels. Such additives 20 acid methyl ester. Transesterification is the preferred method effect the reduction of serum cholesterol through the dis­ to those skilled in the art. ruption of intestinal absorption of dietary cholesterol by A better understanding of the present invention, its several displacing it from bile and micelli. Free sterols or stanols, aspects, and its advantages will become apparent to those though, are not optimum candidates for use in typical skilled in the art from the following detailed description, pharmaceutical or dietary dosage forms as cholesterol reduc- 25 wherein there is shown and described the preferred embodi­ ing agents due to their very high melting points 130 C. and ment of the invention, simply by way of illustration of the low solubility in aqueous and oil media. As a result such best mode contemplated for carrying out the invention. compounds are preferred to be converted into their fatty esters for food applications, which reduce their melting DETAILED DESCRIPTION OF THE points and solubility in oil. However, the fatty acids attached 30 PREFERRED EMBODIMENT to sterol in the current commercial products are from veg­ As used herein the term "sterol ester" includes both the etable oil such as sunflower, canola, or soybean oil. Those plant sterol ester per se as well as the hydrogenated sterol fatty acids provide no pharmaceutical or nutraceutical func­ products which are referred to as stanol and campestanol. tions except increasing the total calories of the products. Such compounds have the following general formula: 35 Conjugated fatty acids are known to have many health Ac---C0---0-ST benefits such as reducing body fat, inhibiting tumor growth and reducing atherosclerosis. Such conjugated fatty acids wherein Ac-CO is an acyl group from a conjugated fatty are naturally found in beef and dairy fats in trace amounts acid and O-ST is a steryl group derived from a sterol/ (0.2-30 mg/g food). One such conjugated fatty acid is stanol. conjugated linoleic acid ( octadecadienoic acid), hereinafter 40 The term "conjugated fatty acid" is intended to refer to referred to as CLA. Cattle convert the linoleic acid in grass conjugated linoleic acid (CLA) which in turn refers to a into CLA by their special digestive processes. However, group of geometrical and positional isomers of linoleic acids since humans cannot produce such conjugated fatty acids, including but not limited to 9,11-octadecadienoic acid, and such additives to the human system must be through the diet. 10---12 octadecadienoic acid. The cis-9, trans-11 isomer is Thus the providing of CLA in a form to permit its use in 45 the most dominant isomer of CLA in dairy products and is dietetic foods would serve as a significant contribution to the also the most biologically active form as known at present. The term "sterol" or "sterol/stanol" as used herein is field of dietetic foods since it would enable the recipient to receive a valuable additive since it is known that CLA is intended to mean the sterol compound per se or its hydro­ effective in increasing body protein or preventing the loss of genated form including stanol and campestanol. body protein in a human, increasing food efficiency in 50 The present invention is based upon my discovery that humans and assists in reducing body fat. through the use of the conjugated fatty acid-CLA-in the esterification of a sterol there is obtained a product which is It is thus an object of the present invention to provide a liquid at ambient temperature and which product has lower novel ester composition consisting essentially of phytoster­ total calories and which product provides the combined ols including plant sterols/stanols and conjugated linoleic 55 benefits of cholesterol control agent and an anticarcinogen acids. and fat reducing agent. Another object of this invention is to prepare sterol and The present invention provides a process for esterfying stanol esters of CLA for their utilization in food and dietary stanols or/and sterols with CLA. This esterification reaction supplement products. may be accomplished either through the reaction of the Another object of the present invention is to provide a 60 sterol with CLA using a esterification catalyst such as process for the production of sterol esters of conjugated sulphonic acids and tin chloride or though the reaction of the linoleic acids through transesterification and/or esterifica­ sterol with CLA methyl ester using a transesterification tion. catalyst such as sodium methoxide and hydroxide. The results of those esterification reactions is a sterol ester of SUMMARY OF THE INVENTION 65 CLA or a stanol ester of CLA. In accordance with one embodiment of the present While any stanol or sterol that is functionalized with a invention, I have discovered that through the esterification hydroxy group is suitable for transesterification and esteri- US 6,413,571 Bl 3 4 fication by the processes as described herein, in one pres­ from the group consisting of cis-9, trans-11-conjugated ently preferred embodiment of the present invention there is linoleic acid and trans-10, cis 12-conjugated linoleic acid. utilized a sterol/stanol selected form the group consisting of The acid-catalyzed esterification reaction of sterol with beta-sitosterol, , and sitostanol. CLA and base-catalyzed transesterification reaction of sterol with CLA methyl ester, respectively, are depicted below Other suitable sterols include but not limited to 5 demonstrating the formation of a sterol ester of CLA per the , , alpha-spinasterol and . present invention. As shown in the reaction mechanism on It is understood that those sterols/stanols for esterifying may the left sterol is reacted with CLA in the presence of an acid be used in pure form or mixed in certain ratios. catalyst to produce sterol ester of CLA. In the reaction Likewise while any isomer of a conjugated linoleic acid mechanism on the right (which represents the preferred is suitable for esterification by the process as described 10 mechanism), sterol is reacted with CLA methyl ester to herein, in one presently preferred embodiment of the present produce sterol ester of CLA in the presence of a base invention there is utilized a conjugated linoleic acid selected catalyst. (R-alkyl or alkenyl groups)

Phytosterol

HO

0 Ul 0 CH3 H3C 0 ~ OH H3C/ cis 9, trans 11-conjugated linoleic acid cis 9, trans 11-conjugated linoleic acid methyl ester

e VJ 0\ ~ wf--' Vi --...) f--' Cd f--'

H3C

cis 9, trans 11-linoleic acid phytosterol ester

O'I US 6,413,571 Bl 7 8 R is defined as following alkyl or alkenyl groups: the reaction was continued under a high vacuum up to 0.01 beta-Sitosterol: -CH( CH3)CH2CH2CH( C2H5)CH mm Hg and the temperature was raised gradually to 110 C. (CH3)2 The reaction continued until no methanol was bubbling, then the mixture was cooled down to about 60 C. before breaking Stigmasterol: -CH(CH3)CH=CHCH(C2H5)CH the vacuum with N2 . 6 grams of warm water (40-50 C.) was (CH3)2 5 added to destroy the catalyst. The mixture was stirred for Campesterol: -CH(CH3)CH=CHCH(CH3)CH(CH3)2 about 1 minute until appearing homogenous and then cen­ (no double bond at 5, 6) trifuged at 5000 G for 5 minutes. The top layer containing Brassicasterol: -CH(CH3)CH=CHCH2CH(CH3)2 sterol esters was collected and washed with 12 g warm water. The mixture was then centrifuged to recover the top Avenasterol: -CH(CH3)CH2CH2C(=CH-CH3)CH 10 sterol ester layer. The sterol ester was then purified by (CH3)2 (double bond at 5, 6 or 7, 8 only) vacuum distillation to remove moisture and residual methyl alpha-Spinasterol: -CH(CH3)CH=CHCH2C(C2H5) esters. The product is liquid at ambient temperature and has CH(CH3)2(double bond at 7, 8) three melting peaks at 15, 37, and 58 C. as measured by Ergosterol: -CH(CH3)CH=CHCH(CH3)CH(CH3)2 DSC. 15 (double bonds at 5, 6 and 7, 8) EXAMPLE 2 A similar reaction system is carried out when the hydro­ To Prepare Sterol Esters from Conjugated Linoleic Acid genated sterol such as stanol is the reactant. CLA One TM, a commercial CLA product available from The molar ratios of the starting materials for the transes­ Pharmanutrients, Inc. and which contains 75% of free fatty terification and esterification reactions are provided in sto­ acid, was used in this synthesis. CLA One TM typically ichiometric levels. It is preferred that the CLA be present in 20 contains with 35% cis 9, trans 11 and 36% trans 10, cis at least 5-10% excess so as to react with all of the sterol or 12-linoleic acids. 150 g of CLA One TM was mixed with 600 stanol. Any excess unreacted CLA is easily removed in the mL methanol and 12 mL concentrated sulfuric acid. The product work-up. mixture was refluxed for 30 minutes to prepare the methyl The usage of esterification catalyst varies with the catalyst esters of fatty acids. The product was washed twice with 100 used and their uses are reviewed in Bailey's Industrial Oil 25 mL 5% sodium chloride and with 2% potassium bicarbonate and Fat Products, 4th edition, edited by Daniel Swem, until nutral pH in the aqueous phase. The methyl esters of Volume 2, PP 113-127. Since esterification involves high fatty acids were dried by heating at 90-105 C. under up to reaction temperature and low reaction rate, sterol ester of 20 mm Hg vacuum. One hundred grams of methyl ester CLA are preferred to be prepared via transesterification. produced as above was mixed with 60 g plant sterols that In carrying out the process of the present invention 30 contains 40% beta-sitosterol, 20-30% campesterol and solvents such as ethers and short chain alkanes may be added 10---30% dihydrobrassicasterol. The mixture was dried at to the reaction mixture to promote reaction. 90---105 C. under up to 20 mm Hg vacuum for about an hour. The reaction rate of transesterification increases at an After cooling the mixture down below 70 C., 0.5 grams of elevated temperature. The typical reaction temperature NaOCH3 powder was added to the reactant mixture as ranges from 40 C. to about 250 C. The reaction period may 35 transesterification catalyst. Vacuum was applied slowly to vary widely, but as a general practice a reaction time in the remove the methanol produced so the reaction proceeded to range of about 4 to about 20 hours can be utilized. The the direction forming sterol esters. When vigorous bubbling reaction is normally carried out for a time which will permit ceased, the reaction was continued under a high vacuum up the reaction to go to completion so that the sterol or stanol to 0.01 mm Hg and the temperature was raised gradually to present is completly esterified. Normally the ester product is 40 110 C. The reaction continued until no methanol was bub­ obtained in yields of greater than 95%. bling out. The mixture was cooled down to about 60 C. and Following completing of the reactions, the resulting ester nitrogen was introduced to break the vacuum. 6 grams of product can be isolated with or without organic solvent 50% citric acid aqueous solution was added to neutralize the extraction after removing the catalyst such as by water transesterification catalyst. The mixture was stirred for about washing. Typical solvents are low boiling point organic 45 6 minutes or until appearing homogenous and then centri­ compounds including but not limited to diethyl or petroleum fuged at 4000 G for 5-30 minutes. The top layer containing ethers, hexane, dichloromethance, chloroform, and toluene. sterol esters was collected and washed twice with 12 g warm The following examples are intended to be illustrative of water. The mixture was then centrifuged at 4000 G for 5 the present invention and to teach one of ordinary skill in the minutes to recover the top sterol ester layer. The sterol ester art to make and use the invention. These examples are not 50 was then purified by vacuum distillation to remove moisture intended to limit the invention in any way. and residual methyl esters. The specific examples herein disclosed are to be consid­ EXAMPLE 1 ered as being primarily illustrative. Various changes beyond To Prepare Sterol Esters from Conjugated Linoleic Acid those described will not doubt occur to those skilled in the Methyl Ester 55 art and such changes are to be understood as forming a part A commercial CLA methyl ester product was used in the of this invention insofar as they fall within the spirit and synthesis, which contains 41 % of cis 9, trans 11, 44% of scope of the appended claims. trans 10, cis 12, and 10% of cis 10, cis 12 conjugated linoleic The inventive compositions are usable as a component in acids. 60 grams of plant sterols containing 40% beta­ any number of food products or as a dietary supplement sitosterol, 20---30% campesterol and 10-30% dihydrobrassi- 60 whereby the compositions may be delivered in a convenient casterol was mixed with 100 grams of CLA methyl ester. form and the advantages thereof may be easily obtained. The mixture is solid at room temperature. After dried at What is claimed is: 90-105 C. under about 20 mm Hg vacuum for about an hour, 1. A process for the production of sterol or stanol esters of the mixture was cooled down to about 70 C., and 1.3 grams a conjugated linoleic acid which comprises reacting under of 25% NaOCH3-Methanol solution was added. A vacuum 65 conditions suitable for effecting transesterification an alco­ of up to 20 mm Hg was applied slowly to remove the hol ester of a conjugated linoleic acid and a sterol or stanol methanol produced. When no vigorous bubbles came out, in the presence of a transesterification catalyst. US 6,413,571 Bl 9 10 2. A process in accordance with claim 1 wherein said 6. A process in accordance with claim 5 wherein said conjugated linoleic acid is selected from the group consist­ sterol is beta-sitosterol. ing of cis-9, trans-11-conjugated linoleic acid and trans-10, 7. A process in accordance with claim 5 wherein said cis-12-conjugated linoleic acid. sterol is campesterol. 3. A process in accordance with claim 2 wherein said 5 8. A process in accordance with claim 5 wherein said conjugated linoleic acid is cis-9, trans-11 conjugated linoleic sterol is stigmasterol. acid. 4. A process in accordance with claim 3 wherein said 9. A process in accordance with claim 5 wherein said conjugated linoleic acid is trans-10, cis-12-conjugated sterol is sitostanol. linoleic acid. 10 10. A process in accordance with claim 5 wherein said 5. A process in accordance with claim 1 wherein said sterol is campestanol. sterol is selected from the group consisting of beta­ sitosterol, campesterol, stigmasterol, sitostanol, and campestanol. * * * * *